RNA molecules, like proteins, fold into specific three-dimensional structures that are required for their biological function. However, our understanding of how RNA molecules attain their native conformations is in its infancy. This Program Project proposes to investigate the mechanism of RNA folding through the integrated application of approaches that each report unique and complementary aspects of the folding reaction, including single molecule fluorescence and force measurements, time-resolved small angle x-ray scattering, time-resolved x-ray "footprinting", computation and enzymology. This arsenal of approaches will be focused on the folding of a single RNA, a ribozyme derived from the self-splicing group I intron of Tetrahymena thermophila. The Tetrahymena ribozyme is the best-understood ribozyme in terms of its structure, folding and catalytic mechanism, and its large size (approximately 400 nucleotides) provides ample three-dimensional structural complexity for investigation of fundamental features of RNA tertiary structure formation. The proposed studies have extensive synergy in their reporting of global and local structure, single molecule and ensemble averages, and will further develop the Tetrahymena ribozyme as a paradigm for future studies comparing and contrasting the behavior of other RNA molecules. The information obtained from this Program will also allow comparison with the folding behavior of proteins, revealing which properties are specific to each class of macromolecule and which are common to both; the common properties may be fundamental to macromolecules that adopt specific biologically active structures. An understanding of the fundamental behavior of RNA provides a starting point for determining how its behavior may be altered, controlled, or augmented by cellular interactions and in therapeutic intervention. Knowledge of these fundamental properties will provide a foundation for studies focusing on the cellular behavior of RNA, on the role of RNA in disease, and on the potential use of RNA as a drug target or therapeutic.